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The ultraviolet circular dichroism of muscle proteins
164 7 8 9 io II I2
H. A. H. P. F. H.
SHORT COMMUNICATIONS F. CHENG AND B. STEINBERG, J. Lab. Clin. Med., 63 (1964) 694. H. GORDON, Biochim. Biophys. Acta, 42 (196o) 23. E. SCHULTZE,Clin. Chira. Acta, 3 (1958) 24. FIREMAN, W. E. VANNIER AND H. C. GOODMAN,Proc. Soc. Exptl. Biol. ;!/ied., 115 (1964) 845. C. GREENWOOD, W. Yl. HUNTER AND J. S. GLOVER, Biochem. J., 89 (1963) 114. J. GILBERMANN, Lancet, ii (1957) 26.
Received May 2nd, 1968 Biochim. Biophys. Acta, 168 (1968) 161-164
BBA 33104 T h e u l t r a v i o l e t c i r c u l a r d i c h r o i s m of muscle p r o t e i n s
The present communication reports the results of an ultraviolet circular dtchroism (CD) examination of a series of muscle proteins, which yield in the condensed state the a-type wide-angle X-ray diffraction pattern. Three members of this class (tropomyosin, light meromyosin fraction I and paramyosin) are similar in molecular architecture, in that hydrodynamic, X-ray and optical rotatory dispersion (ORD) measurements suggest that they are made up of 2 a-helices arranged side by side and twisted about one another z-3. As such their native molecules function as excellent prototypes for defining the CD characteristics of the protein a-helical form. In addition, CD spectra are presented for the two enzymatically active, but lower helix containing muscle proteins, heavy meromyosin and its parent, myosin. It should be added that MOMMAERTS4 has reported the ultraviolet CD characteristics of myosin, as measured in a Durrum-Jasco CD spectrophotometer. The CD measurements were made on a Cary model 6oo1 CD attachment to a Cary 60 recording spectropolarimeter, equipped with a water cooled lamp housing maintained at 27 °. The instrument was calibrated with an aqueous solution of (d)-IOcamphor sulfonic acid (Eastman Organic Chemicals, recrystallized) with a difference in molecular absorbance coefficient (eL -- eR) of 2.16. Constant nitrogen flushing was employed over the wavelength range examined (185-25o m/~). All measurements were made in o.5-mm cells, and protein concentrations were in the range o.o2-o.o6%. The mean residue molecular ellipticity obtained is given by: OOM
[O] ioo
lc
where M is the mean residue molecular weight (here taken as 115 for all the proteins), O ° is the observed ellipticity in degrees, I is the cell pathlength in dm and c is the protein concentration in g/cm ~. The units of [O] are degrees-cm ~ per dmole. Representative CD spectra obtained for the various muscle protein systems studied are shown in Fig. I. All 5 proteins exhibit two negative dichroic peaks located at 222 and 2o 9 m/~, with a crossover point at 2oi-2o2 m#, and there is also present a single positive dichroic band at 191 m# (inset, Fig. I). These spectra and their characA b b r e v i a t i o n s : CD, circular d i c h r o i s m O R D , optical r o t a t o r y dispersion.
Biochim. Biophys. Acta, i68 (1968) I 6 4 - I 6 7
L
E"
(a) o.6 M K C l - o . o i M p h o s p h a t e (b) o.i M p h o s p h a t e
Venus mercenaria p a r a m y o s i n
5oo
900
500
--39 800 4- 3900 39 7 °0
--18 360 4-
--23 250 ± 14oo
--38 94 ° 4-
--38 9 4 o i
--44 45 ° ± 13o°
[ 0 ] ~ ml~
POLYPEPTIDES
5oo
900
50o
--38 900 4- 3900 --37 ooo
--17 590 4-
--21 75 ° 4- 14oo
--36 3oo 4-
--36 9 4 0 ±
--41 290 4- 13oo
[ O ] 1 0 9 m/~
4-71 500 4- 61oo 4-82 4o0
+ 3 7 020 ± 2300
+ 4 8 44 ° 4- 2500
4-87 ooo 4- 3ooo
+8768o±3ooo
+ 9 8 47 ° 4- 3000
[ O ] 1 9 1 m/*
* D a t a of TOWNEND el al. ~. ** D a t a of CASSIM AND YANG8 obtained f r o m their Fig. i b y m e a s u r i n g a m p l i t u d e s of b a n d ellipticities in a Nikon m i c r o c o m p a r a t o r .
H20 (pH 11.2) o.i M NaC104 (pH 4.5)
(a) H20 (b) 0. 5 IV[N H 4 F
R a b b i t skeletal h e a v y meromyosin
Polylysine* Poly-L-glutamic acid**
(a) 0. 5 M KCl-o.o 5 M p h o s p h a t e (b) 0.5 M N H 4 F
R a b b i t skeletal myosin
KCl-o.oi M phosphate NH4F phosphate phosphate
(a) (b) (c) (d)
R a b b i t skeletal t r o p o m y o s i n
M M M M
(a) 0.6 M K C l - o . o i M p h o s p h a t e (b) o. 5 M NH~F
R a b b i t skeletal light meromyosin fraction I
0.6 0.5 0. 3 o.I
Solvent systems
Polymer
AND REFERENCE
OF MUSCLE PROTEINS
CIRCULAR DICHROISM PARAMETERS
TABLE I
Ln
c~ o
o
I~6
SHORT COMMUNICATIONS
teristics correspond closely to those determined for several helical polypeptide@, ~, and all 3 bands are due to spectral transitions associated with peptide bands arranged in right-handed helical array. The absolute values of the ellipticity at the extrema at 222, 2o 9 and 191 m# are recorded in Table I for the protein systems, as well as for two reference helical polypeptides, polylysine and poly-L-glutamic acid. No attempt has been made to calculate helical content from this data, since the reference ellipticity values for high molecular weight helical polymers vary considerablyT, s. However, it is apparent that the three coiled-coil proteins (light meromyosin fraction I, tropomyosin and paramyosin) possess ellipticity bands of comparable intensity to that of the reference a-helical polymers, and by analogy, must therefore be essentially completely a-helical. On the
i
lo-
ii
,.
~O x
0 I
II
180
X in
20C rn~
200
220 in rn~
240
Fig. I. Circular dichroism of fibrous muscle proteins over the wavelength range I85-25o mff, O - - O , myosin; D - - D , heavy meromyosin; Q---O, paramyosin; II--II, tropomyosin; & - - A , light meromyosin fraction I.
other hand, with myosin and heavy meromyosin, the ratios of their ellipticities at the 3 extrema to that of lOO% a-helical poly-L-glutamic acid is 58-60% and 45-48%, respectively. In addition, our data for myosin agree reasonably well, in terms of position and amplitude of ellipticity bands, with that of MOMMAERTS4. It is to be noted that these conclusions regarding relative helicity are in good agreement with estimates of a-helical content deduced for these fibrous muscle proteins by the sister technique of ORD, in the visible and ultraviolet regions of the spectrumg, 1°. The authors wish to acknowledge the invaluable technical assistance of A. KERI and V. LEDSHAM during the course of this work. They are also indebted for financial support to the Canadian Muscular Dystrophy Association, the Medical Research Biochim. Bioph),s. dcta, 168 (1968) I64-~67
167
SHORT COMMUNICATIONS
Council of Canada, the Life Insurance Medical Research Fund and the National Institutes of Health (AM-o6287).
Department o/Biochemistry, University of Alberta, Edmonton, Alberta (Canada) I 2 3 4 5 6 7 8 9
IO
K . OIKAWA C. M. K A Y
W. D. McCuBBIN
S. LOWEY, J. KUCERA AND A. HOLTZER, J. Mol. Biol., 7 (1963) 234. C. COHEN AND K. C. HOLMES, J. Mol. Biol., 6 (1963) 423 . W. D. McCuBBIN, R. F. KOUBA AND C. M. KAY, Biochemistry, 6 (1967) 2417. W. F. H. M. MOMMAERTS, J. Mol. Biol., 15 (1966) 377G. HOLZWARTH AND P. DOTY, J. Am. Chem. Soc., 87 (1965) 218. R. TOWNEND, T. F. KUMOSINSKI, S. N. TIMASHEFF, G. ~). FASMAN AND B. DAVIDSON, Biochem. Biophys. Res. Commun., 23 (1966) 163. S, BEYCHOK, Science, 154 (1966) 1288. J. Y. CASSIM AND J. T. YANG, Biochem. Biophys. Res. Commun., 26 (1967) 58. C. COHEN AND A. G. SZENT-GY(~RGYI, J. Am. Chem., Soc., 79 (1957) 248. ~NT.S. SIMMONS, C. COHEN, A. G. SZENT-GYORGYI, D. B. WETLAUFER AND E. R. BLOUT, J. Am. Chem. Soc., 83 (1961) 4766.
Received May 29th, 1968 Biochim. Biophys. Acta, 168 (1968) 164 167
H. A. H. P. F. H.
SHORT COMMUNICATIONS F. CHENG AND B. STEINBERG, J. Lab. Clin. Med., 63 (1964) 694. H. GORDON, Biochim. Biophys. Acta, 42 (196o) 23. E. SCHULTZE,Clin. Chira. Acta, 3 (1958) 24. FIREMAN, W. E. VANNIER AND H. C. GOODMAN,Proc. Soc. Exptl. Biol. ;!/ied., 115 (1964) 845. C. GREENWOOD, W. Yl. HUNTER AND J. S. GLOVER, Biochem. J., 89 (1963) 114. J. GILBERMANN, Lancet, ii (1957) 26.
Received May 2nd, 1968 Biochim. Biophys. Acta, 168 (1968) 161-164
BBA 33104 T h e u l t r a v i o l e t c i r c u l a r d i c h r o i s m of muscle p r o t e i n s
The present communication reports the results of an ultraviolet circular dtchroism (CD) examination of a series of muscle proteins, which yield in the condensed state the a-type wide-angle X-ray diffraction pattern. Three members of this class (tropomyosin, light meromyosin fraction I and paramyosin) are similar in molecular architecture, in that hydrodynamic, X-ray and optical rotatory dispersion (ORD) measurements suggest that they are made up of 2 a-helices arranged side by side and twisted about one another z-3. As such their native molecules function as excellent prototypes for defining the CD characteristics of the protein a-helical form. In addition, CD spectra are presented for the two enzymatically active, but lower helix containing muscle proteins, heavy meromyosin and its parent, myosin. It should be added that MOMMAERTS4 has reported the ultraviolet CD characteristics of myosin, as measured in a Durrum-Jasco CD spectrophotometer. The CD measurements were made on a Cary model 6oo1 CD attachment to a Cary 60 recording spectropolarimeter, equipped with a water cooled lamp housing maintained at 27 °. The instrument was calibrated with an aqueous solution of (d)-IOcamphor sulfonic acid (Eastman Organic Chemicals, recrystallized) with a difference in molecular absorbance coefficient (eL -- eR) of 2.16. Constant nitrogen flushing was employed over the wavelength range examined (185-25o m/~). All measurements were made in o.5-mm cells, and protein concentrations were in the range o.o2-o.o6%. The mean residue molecular ellipticity obtained is given by: OOM
[O] ioo
lc
where M is the mean residue molecular weight (here taken as 115 for all the proteins), O ° is the observed ellipticity in degrees, I is the cell pathlength in dm and c is the protein concentration in g/cm ~. The units of [O] are degrees-cm ~ per dmole. Representative CD spectra obtained for the various muscle protein systems studied are shown in Fig. I. All 5 proteins exhibit two negative dichroic peaks located at 222 and 2o 9 m/~, with a crossover point at 2oi-2o2 m#, and there is also present a single positive dichroic band at 191 m# (inset, Fig. I). These spectra and their characA b b r e v i a t i o n s : CD, circular d i c h r o i s m O R D , optical r o t a t o r y dispersion.
Biochim. Biophys. Acta, i68 (1968) I 6 4 - I 6 7
L
E"
(a) o.6 M K C l - o . o i M p h o s p h a t e (b) o.i M p h o s p h a t e
Venus mercenaria p a r a m y o s i n
5oo
900
500
--39 800 4- 3900 39 7 °0
--18 360 4-
--23 250 ± 14oo
--38 94 ° 4-
--38 9 4 o i
--44 45 ° ± 13o°
[ 0 ] ~ ml~
POLYPEPTIDES
5oo
900
50o
--38 900 4- 3900 --37 ooo
--17 590 4-
--21 75 ° 4- 14oo
--36 3oo 4-
--36 9 4 0 ±
--41 290 4- 13oo
[ O ] 1 0 9 m/~
4-71 500 4- 61oo 4-82 4o0
+ 3 7 020 ± 2300
+ 4 8 44 ° 4- 2500
4-87 ooo 4- 3ooo
+8768o±3ooo
+ 9 8 47 ° 4- 3000
[ O ] 1 9 1 m/*
* D a t a of TOWNEND el al. ~. ** D a t a of CASSIM AND YANG8 obtained f r o m their Fig. i b y m e a s u r i n g a m p l i t u d e s of b a n d ellipticities in a Nikon m i c r o c o m p a r a t o r .
H20 (pH 11.2) o.i M NaC104 (pH 4.5)
(a) H20 (b) 0. 5 IV[N H 4 F
R a b b i t skeletal h e a v y meromyosin
Polylysine* Poly-L-glutamic acid**
(a) 0. 5 M KCl-o.o 5 M p h o s p h a t e (b) 0.5 M N H 4 F
R a b b i t skeletal myosin
KCl-o.oi M phosphate NH4F phosphate phosphate
(a) (b) (c) (d)
R a b b i t skeletal t r o p o m y o s i n
M M M M
(a) 0.6 M K C l - o . o i M p h o s p h a t e (b) o. 5 M NH~F
R a b b i t skeletal light meromyosin fraction I
0.6 0.5 0. 3 o.I
Solvent systems
Polymer
AND REFERENCE
OF MUSCLE PROTEINS
CIRCULAR DICHROISM PARAMETERS
TABLE I
Ln
c~ o
o
I~6
SHORT COMMUNICATIONS
teristics correspond closely to those determined for several helical polypeptide@, ~, and all 3 bands are due to spectral transitions associated with peptide bands arranged in right-handed helical array. The absolute values of the ellipticity at the extrema at 222, 2o 9 and 191 m# are recorded in Table I for the protein systems, as well as for two reference helical polypeptides, polylysine and poly-L-glutamic acid. No attempt has been made to calculate helical content from this data, since the reference ellipticity values for high molecular weight helical polymers vary considerablyT, s. However, it is apparent that the three coiled-coil proteins (light meromyosin fraction I, tropomyosin and paramyosin) possess ellipticity bands of comparable intensity to that of the reference a-helical polymers, and by analogy, must therefore be essentially completely a-helical. On the
i
lo-
ii
,.
~O x
0 I
II
180
X in
20C rn~
200
220 in rn~
240
Fig. I. Circular dichroism of fibrous muscle proteins over the wavelength range I85-25o mff, O - - O , myosin; D - - D , heavy meromyosin; Q---O, paramyosin; II--II, tropomyosin; & - - A , light meromyosin fraction I.
other hand, with myosin and heavy meromyosin, the ratios of their ellipticities at the 3 extrema to that of lOO% a-helical poly-L-glutamic acid is 58-60% and 45-48%, respectively. In addition, our data for myosin agree reasonably well, in terms of position and amplitude of ellipticity bands, with that of MOMMAERTS4. It is to be noted that these conclusions regarding relative helicity are in good agreement with estimates of a-helical content deduced for these fibrous muscle proteins by the sister technique of ORD, in the visible and ultraviolet regions of the spectrumg, 1°. The authors wish to acknowledge the invaluable technical assistance of A. KERI and V. LEDSHAM during the course of this work. They are also indebted for financial support to the Canadian Muscular Dystrophy Association, the Medical Research Biochim. Bioph),s. dcta, 168 (1968) I64-~67
167
SHORT COMMUNICATIONS
Council of Canada, the Life Insurance Medical Research Fund and the National Institutes of Health (AM-o6287).
Department o/Biochemistry, University of Alberta, Edmonton, Alberta (Canada) I 2 3 4 5 6 7 8 9
IO
K . OIKAWA C. M. K A Y
W. D. McCuBBIN
S. LOWEY, J. KUCERA AND A. HOLTZER, J. Mol. Biol., 7 (1963) 234. C. COHEN AND K. C. HOLMES, J. Mol. Biol., 6 (1963) 423 . W. D. McCuBBIN, R. F. KOUBA AND C. M. KAY, Biochemistry, 6 (1967) 2417. W. F. H. M. MOMMAERTS, J. Mol. Biol., 15 (1966) 377G. HOLZWARTH AND P. DOTY, J. Am. Chem. Soc., 87 (1965) 218. R. TOWNEND, T. F. KUMOSINSKI, S. N. TIMASHEFF, G. ~). FASMAN AND B. DAVIDSON, Biochem. Biophys. Res. Commun., 23 (1966) 163. S, BEYCHOK, Science, 154 (1966) 1288. J. Y. CASSIM AND J. T. YANG, Biochem. Biophys. Res. Commun., 26 (1967) 58. C. COHEN AND A. G. SZENT-GY(~RGYI, J. Am. Chem., Soc., 79 (1957) 248. ~NT.S. SIMMONS, C. COHEN, A. G. SZENT-GYORGYI, D. B. WETLAUFER AND E. R. BLOUT, J. Am. Chem. Soc., 83 (1961) 4766.
Received May 29th, 1968 Biochim. Biophys. Acta, 168 (1968) 164 167